The retrotrapezoid nucleus (RTN) is a region critical for respiratory chemoreception. This region contains a subset of neurons that are intrinsically sensitive to changes in CO2/H+ and communicate this information to other respiratory centers to regulate depth and frequency of breathing. Interestingly, RTN astrocytes also appear to be specialized to support the activity of chemosensitive neurons by providing a CO2/H+-dependent purinergic drive that directly enhances activity of chemosensitive neurons. Pharmacological evidence suggests that the mechanism by which RTN astrocytes sense CO2/H+ involves inhibition of Kir4.1-5.1 channels, therefore, to definitively test the role of Kir4.1 and astrocyte in respiratory control, we made an astrocyte specific inducible Kir4.1 knockout mouse model (Kir4.1 cKO). We found at the cellular level that astrocytes in slices from Kir4.1 cKO mice no longer express a CO2/H+-sensitive current or modulate chemosensitive neurons by a purinergic-dependent mechanism. At the whole animal level, we found that Kir4.1 cKO mice show a reduced ventilatory response to CO2. It is also well known that cerebral blood flow is highly sensitive to changes in CO2/H+; an increase in CO2/H+ will cause vasodilation and increased blood flow, which in turn will facilitate removal of excess CO2/H+. Considering that CO2/H+-induced vasodilation would counter-regulate chemoreceptor activity, we hypothesize that CO2/H+-evoked ATP release from astrocytes will antagonize CO2/H+-vasodilation in the RTN, and thus prevent CO2/H+ washout and further enhance chemoreceptor function. Therefore the second goal of this study is to determine whether purinergic signaling in the RTN provides specialized control of vascular tone in a manner that contributes to the drive to breathe. We show in vitro and in anesthetized rats that purinergic signaling in the RTN maintains vascular tone during high CO2/H+, and disruption of this mechanism by P2 receptor blockade with PPADS (100 µM) decreased the ventilatory response to CO2. Together, these results expand our understanding of how RTN astrocytes control breathing by showing that i) CO2/H+-evoked ATP release contributes to chemoreception by preventing CO2/H+-induced vasodilation; and ii) Kir4.1 is an important component of astrocyte chemoreception.